Exposure of the nematode Caenorhabditis elegans to a heat shock results in the induction of a number of genes not normally expressed in the animals under normal growth conditions. Among these are a family of genes encoding 16 kDa heat shock proteins (hsp16s). The major hsp16 genes have been cloned and characterized, and found to reside at two clusters in the C. elegans genome. One cluster contains two distinct genes, hsp16-1 and hsp16-48, arranged in divergent orientations separated by only 348 base pairs (bp). An identical pair, duplicated and inverted with respect to the first pair, is located 415 bp away. This cluster, located on chromosome V, therefore contains four genes as two identical pairs within less than 4 kilobases of DNA, and the pairs form the arms of a large inverted repeat. A second pair of genes, hsp16-2 and hsp16-41, constitutes a second hsp16 locus with an organization very similar to that of the hsp16-1/48 locus, except that it is not duplicated. Comparisons of the derived amino acid sequences show that hsp16-1 and hsp16-2 form a closely related pair, as do hsp16-41 and hsp16-48. These hsps show extensive sequence identity with the small hsps of Drosophila, as well as with mammalian alpha-crystallins. The coding region of each gene is interrupted by a single intron of approximately 50 bp, in a position homologous to that of the first intron in a mouse alpha-crystallin gene. The compact intergenic regions of both hsp16 loci contain a TATA element and a heat shock element (HSE) for each member of the pair, and are very similar in sequence overall. Expression studies, however, show that the level of transcripts from the hsp16-2/41 pair may be up to 14-fold higher on a per gene basis, as the level of RNA from the hsp16-1/48 pair, depending upon the induction conditions and developmental stage. This difference in message levels seems to be due to differences in the kinetics of inactivation of the genes rather than in transcription rates or rates of mRNA turnover. Distinct DNAseI hypersensitive sites are present upstream of each HSE in chromatin when the genes are inactive; these disappear and the whole intergenic region seems to become DNAse sensitive when the genes are maximally active.Key words: heat shock, 16-kDa polypeptides, gene structure, transcription, DNAseI hypersensitive sites, Caenorhabditis elegans.